1. Field of the Invention
The present invention generally relates to devices and methods for rapidly identifying chemicals with biological activity in liquid samples, particularly automated screening of low volume samples for new medicines, agrochemicals, or cosmetics.
2. Description of the Related Art
Drug discovery is a highly time dependent and critical process in which significant improvements in methodology can dramatically improve the pace at which a useful chemical becomes a validated lead, and ultimately forms the basis for the development of a drug. In many cases the eventual value of a useful drug is set by the timing of its arrival into the market place, and the length of time the drug enjoys as an exclusive treatment for a specific ailment.
A major challenge for pharmaceutical companies is to increase the speed and efficiency of this process while at the same time minimizing costs. One solution to this problem has been to develop high throughput screening systems that enable the rapid analysis of many thousands of chemical compounds per 24 hours. To reduce the otherwise prohibitive costs of screening such large numbers of compounds, typically these systems use miniaturized assay systems that dramatically reduce reagent costs, and improve productivity. To efficiently handle large numbers of miniaturized assays it is necessary to implement automatic robotically controlled analysis systems that can provide reliable reagent addition and manipulations. Preferably these systems and the invention herein are capable of interacting in a coordinated fashion with other systems sub-components, such as a central compound store to enable rapid and efficient processing of samples.
Miniaturized high throughput screening systems require robust, reliable and reproducible methods of analysis that are sensitive enough to work with small sample sizes. While there are a large number of potential analysis methods that can successfully be used in macroscopic analysis, many of these procedures are not easily miniaturizable, or lack sufficient sensitivity when miniaturized. This is typically true because absolute signal intensity from a given sample decreases as a function of the size of the sample, whereas background optical or detector noise remains more or less constant for large or small samples. Preferred assays for miniaturized high throughput screening assays have high signal to noise ratios for very small sample sizes.
Fluorescence based measurements have high sensitivity and perform well with small samples, where factors such as inner filtering of excitation and emission light are reduced. Fluorescence based measurements therefore exhibit good signal to noise ratios even with small sample sizes. A particularly preferred method of using fluorescence based signal detection is to generate a fluorescent (emission) signal that simultaneously changes at two or more wavelengths. A ratio can be calculated based on the emission light intensity at the first wavelength divided by the emitted light intensity at a second wavelength. This ratiometric measurement of a fluorescent assay has several important advantages over other non-ratiometric types of analysis. Firstly, the ratio is largely independent of the actual concentration of the fluorescent dye that is emitting fluorescence. Secondly, the ratio is largely independent of the intensity of light with which the fluorescent compound is being excited. Thirdly, the ratio is largely independent of changes in the sensitivity of the detector, provided that is that these changes are the same for the detection efficiency at both wavelengths. This combination of advantages makes fluorescence based ratiometric assays highly attractive for high throughput screening systems, where day to day, and, assay to assay reproducibility are important.
Traditionally, there are two general ways to read fluorescence from a multi-well plate. In one arrangement, a read head is moved from well to well and at each well, there is a dwell time during which the fluorescence signal is digitized and stored into memory. Optically, this scheme is the simplest. There is only one optical assembly, one set of filters and one detector. However, depending on how many wells there are in the plate, the read time can be unacceptably long. It is not just the dwell times that contribute to the total read time. Every time the read head is moved from well to well, it takes time to accelerate and decelerate the stage used in moving the setup.
In the other arrangement, some sort of parallelism is employed. Either a picture is taken of the plate, using a CCD camera or some other imaging arrangement, or multiple optical read heads are employed. The advantage of this arrangement is a significant reduction in the read time. However, a new difficulty is introduced, namely that of normalization. When several wells are read at the same time by several read heads, the question that arises is how to make sure that these heads behave in the same way in terms of collection efficiency, detector sensitivity, filter quality and the like. In the case of a CCD camera, the analogous issue is one of flat-fielding. Accordingly, improved methods and systems for rapidly and accurately measuring fluorescence signals in high throughput screening environments are needed.
In one embodiment, a method in accordance with the invention comprises placing a platform on a detector for detecting sample attributes, wherein the detector has an adjustable parameter. The method includes making a plurality of measurements of a characteristic of the platform to obtain a plurality of measured values of the characteristic at different locations on the platform. The attributes are measured with the detector with the parameter adjusted based upon the measured values.
In one embodiment, the platform is a multi-well plate, the characteristic comprises a bottom contour of the multi-well plate, and the adjustable parameter comprises the distance between an optical detector and the multi-well plate bottom. In some of these embodiments, to maintain the optimal distance between the optics and the plate, the detector is provided with a sensor used to detect the location of a plate element of interest. In addition, the optics are mounted on an optics positioning device, for example a linear stage. In one embodiment, the optics positioning device may be oriented in the vertical axis so that the vertical position of the optics may be adjusted relative to the plate. Alternative to adjusting the optics, the plate positioner may be mounted on a stage oriented in the vertical axis and the plate may be moved to adjust the relative position of the plate and optics. The optics positioning device is slaved to the stages on the plate positioner which controls the horizontal position of the plate relative to the optics.
The method of reading a microplate while compensating for plate variations comprises first mapping the topology of the plate element of interest, for example the elevation of the plate bottom relative to the optics. The plate is moved relative to the sensor along all or a representative portion of the plate element of interest. The plate may be moved in any desired pattern which covers the desired area, but is preferably moved in a raster scan. As the plate is moved, the sensor measures the location of the plate element of interest. From these measurements a contour map of the plate element of interest may be computed which describes the plate topology. After the mapping is complete, the scanner reads the plate. During the reading scan, the optics positioning device adjusts the positioning of the optics based upon the mapped plate topology to maintain the desired distance between the optics and the critical plate element. For example, the optics positioning device may adjust the distance between the optics and the bottom of the plate to maintain a substantially constant distance between the optics and the bottom of the plate as each of the wells is scanned. In some advantageous embodiments, the reading scan is performed in an essentially continuous fashion along rows of wells, avoiding dwell time on each well and thereby decreasing the time required to scan a multi-well plate.
In another embodiment, an apparatus for detecting an attribute of a plurality of samples disposed on a platform comprises a platform positioning stage for supporting the platform and for aligning a predetermined location of the platform with a predetermined position. The apparatus further comprises a first sensor which measures a feature of the platform at a plurality of different locations and transmits the measured values to a controller. A detector is provided having a signal output representative of the attribute, wherein the detector is positioned to detect the attribute of a sample when the sample is aligned with the predetermined position, and wherein the detector has optics for receiving light transmitted from the samples and the optics are mounted on an optics positioning device. In addition, a controller is adapted to receive the measured values and to transmit a signal to an optics positioning device to control the position of the optics relative to the platform, wherein the optics positioning device is adapted to automatically adjust the position of the optics relative to the platform in response to a signal from the controller based upon the measured values.
Other embodiments of the invention include methods of identifying a chemical having modulating activity with respect to a target molecule. In one specific embodiment, such a method comprises retrieving a plurality of sample chemicals from a chemical storage and retrieval module, placing the plurality of sample chemicals in respective wells of a multiwell plate; routing the multiwell plate to an automated analysis system comprising a detector adapted to measure an attribute of a chemical, biological or biochemical sample, wherein the detector has a detection head, an adjustable parameter and a sensor for measuring a feature of the multiwell plate. The multi-well plate is placed on the detector, and a plurality of measurements are made with the sensor of the feature at or proximate to a plurality of predetermined locations on the multiwell plate. This particular method additionally comprises measuring, with the detector, an attribute of the samples in each predetermined well with the adjustable parameter adjusted based upon the measured value of the feature at the predetermined location at or proximate to each predetermined well so as to obtain measured attribute values for each sample. The measured attribute values are analyzed to detect the modulating activity.
Furthermore, another embodiment of the invention comprises a medicament made by a process comprising identifying a pharmacologically active chemical by a process including loading chemicals into a plurality of wells of a multiwell plate, placing the multiwell plate in a detector adapted to measure an attribute of a chemical, biological or biochemical sample, the detector having a detection head, an adjustable parameter and a sensor for measuring a feature of the multiwell plate, and making a plurality of measurements with the sensor of the feature at or proximate to a plurality of predetermined locations on the multiwell plate to obtain a measured value of the feature at each of the predetermined locations, each of the predetermined locations being at or proximate to a predetermined well of the multiwell plate. The method also comprises measuring, with the detector, an attribute of the samples in each predetermined well with the adjustable parameter adjusted based upon the measured value of the feature at the predetermined location at or proximate to each predetermined well, thereby detecting pharmacological activity of at least one of the samples and incorporating an effective amount of at least one of the pharmacologically active samples into a biocompatible carrier.